Abstract
Glioblastoma (GBM) is a highly aggressive primary brain tumor with limited treatment options and poor prognosis. Current standard of care therapy includes tumor resection followed by chemotherapy and/or radiotherapy. However, success of these therapies for GBM is challenged by inefficient therapeutic delivery to the tumor due to the blood-brain- (BBB) and blood-tumor-barriers (BTB), and some tumors cannot be resected due to location in the brain. Despite decades of research, patient prognosis remains poor with a median survival of ~16 months, highlighting the need for more targeted therapeutic delivery platforms.
The inefficiency of the current aggressive standard of care treatment strategy has motivated this work to develop more targeted and effective therapeutic approaches for the treatment of GBM. Gene therapy offers potential for GBM by altering the genetic expression and behavior of cells in the tumor microenvironment to generate an anti-tumor response. Systemically administered genetic material must evade the reticuloendothelial system (RES) and efflux transporters expressed in the BBB, pass through the extracellular matrix (ECM) and cell membrane, and escape lysosomal degradation to reach their target. To aid in overcoming these biological barriers and increase circulation time, genetic material can be encapsulated in nanoparticles (NPs). Studies have pursued gene therapy to halt angiogenesis, drive an anti-tumor immune response, reverse drug resistance, and more. However, these therapies suffer from a lack of delivery and transfection efficiency. To overcome drug delivery challenges, focused ultrasound (FUS) can be leveraged to temporarily induce BB/BTB opening and facilitate therapeutic delivery. During FUS treatments, intravenously administered microbubbles oscillate within the FUS focal zone, exerting mechanical forces that safely and temporarily disrupt vasculature. This process enhances vascular permeability, enabling targeted and localized delivery of therapeutic agents directly to the tumor site.
Though FUS can be used to overcome limitations associated with therapeutic delivery, the next challenge we are faced with is therapeutic retention. P-glycoprotein, or P-gp, is a drug efflux transporter that prevents intracellular accumulation of exogenous substances and is expressed by both endothelium in the BB/BTB and cancer cells. While P-gp is an important line of defense for protecting healthy brain from foreign substances, it further hinders therapeutic delivery in the context of GBM. Anti-cancer therapies, such as chemotherapies, that reach target cells are often rapidly effluxed from P-gp-expressing cells back into the bloodstream, allowing only a small fraction of the administered drug to be retained by the tumor. In aim 1, we explore knocking out P-gp to prevent drug efflux by using CRISPR/Cas9 ribonucleoproteins (RNPs). Following direct injection of naked RNPs or systemic administration of RNPs encapsulated in lipid NPs, followed by FUS-mediated opening of the BB/BTB, we observed improved tumor growth control and survival of mice treated with paclitaxel (PTX) chemotherapy. These data demonstrate improved therapeutic efficacy following targeted P-gp knockout in GBM tumors.
Aim 2 of this dissertation focuses on characterizing transfection efficacy and cellular tropism, or specificity, of a novel gene delivery vector. To improve targeting precision, NPs are often modified with surface ligands designed to bind to molecules that are overexpressed by the target tissue. Exofacial thiols, or thiol (SH-) groups located on the cell membrane, are enriched in tumors due to the dysregulated protein synthesis, increased redox activity, elevated pH, and high metabolic activity within the tumor microenvironment (TME). In our research, we modified NPs to contain free thiol groups, thereby creating SH-NPs. These thiol groups are designed to act as “targeting ligands” for exofacial thiols to enhance their accumulation within the tumor. Other groups have explored the use of SH- groups in NP formulations polymer crosslinking to improve NP stability, formation of disulfide bonds to be cleaved in oxidative environments, and to conjugate other targeting ligands or nucleic acids to polymers, however, our study is the first to explore the use of SH- as a targeting functional group for tumors for non-viral gene carriers. In combination with FUS, SH-NPs demonstrated exceptional targeting of the tumor endothelium. We then used this platform to selectively transfect the tumor endothelium with a chemokine-encoding plasmid to improve immune cell recruitment to the tumor.
In summary, these studies investigate the development of innovative gene delivery platforms and the use of FUS to address challenges in therapeutic delivery and drug retention for the enhancement of anti-tumor efficacy of treatments for GBM.
